We are grateful for the instrumentation and laboratory provided byextend our gratitude to the Mongolian medical faculty of the Inner Mongolian Medical University, China.This study was supported by, for providing the necessary instrumentation and laboratory facilities. This study received financial support from the National Natural Sciences Foundation of China (Grant No. 81760762) and), the Inner Mongolia Medical University Project of China (Grant No. YKD2022MS074), and the Scientific Research Project of Higher Education in Inner Mongolia, China (Grant No. NJZY22661) and the Open Fund Project of Key Laboratory of Chinese and Mongolian Medicine in Inner Mongolia Autonomous Region,China (Grant No. MYX2023-K07).

The experimental protocols received approval from the Ethics of Animal Experiment Care Committee of Inner Mongolia Medical University (YKD202301172) and adhered to the National Institutes of Health guidelines for animal care and ethics. The license number for our animal center is NO.110324230102364187. Twenty-four male Sprague-Dawley (SD) rats, each 8 weeks old (200 g &#177; 20 g), were acquired and housed in a controlled environment with a temperature of 22&#176;C &#177; 2&#176;C and humidity of 55% &#177; 15%. Feed the rats with a rodent maintenance-feed diet and pure water with corn cobs for bedding. The rats were subjected to a 12 h/12 h light/dark cycle for 1 week prior to experimentation.
1. Establishment of CUMS rat model
<ol>
	<li>Grouping
	<ol>
		<li>Divide the 24 rats randomly into 2 groups: the control (CON) group, which will not be exposed to isolation or stress, and the model (MOD) group. Each group contains 12 rats.</li>
		<li>House the rats in standard cages measuring 55 cm x 40 cm x 20 cm, with 6 rats per cage. Retain the cage assignment throughout the acclimatization period unless otherwise indicated.</li>
		<li>Fill each rearing cage with fresh bedding and replace it twice a week.</li>
		<li>Conduct a 1-week acclimatization period. Allow the rats unrestricted access to food and water, except during CUMS stressor application. Maintain a constant environment with a temperature of 22&#176;C &#177; 2&#176;C, humidity of 55% &#177; 15%, and a 12 h/12 h light/dark cycle from 08:00 to 20:00, unless otherwise stated.</li>
		<li>Prior to initiating the experiment, handle the rats daily to acclimate them to the researcher and to minimize additional stress during the experimental phase.</li>
	</ol>
	</li>
	<li>Isolation with chronic unpredictable mild stress
	<ol>
		<li>Concurrently place the MOD and CON groups in separate rooms. House the MOD group rats individually, while keeping the CON group rats together. Maintain all other conditions constant.</li>
		<li>Implement a 28-day stressor regimen25. To prevent habituation and to ensure stressor unpredictability, administer one random stressor per day, avoiding the use of the same stressor on consecutive days.</li>
		<li>Randomly apply one of the following nine stressors26,27 on different days: 24 h water deprivation, 24 h food deprivation, wet padding, cage incline, inversion of the light/dark cycle, cold exposure at 4 &#176;C, heat exposure at 45 &#176;C, 1 min tail clamping, or 15 min shaking at 160 rpm. The specific design is outlined in Table 1.</li>
		<li>During the application of the stressor, restrict access to food and water to the MOD group until the stress was over, except during the inversion of the light/dark cycle. The CON group was not required to restrict water and diet.</li>
	</ol>
	</li>
	<li>Stress methods
	<ol>
		<li>Initiate the experiment by applying depression stimuli in conjunction with 28-day isolation to all rats, excluding the control group. House these rats in individual cages. Refer to Table 2 for conditions related to depression stimuli.</li>
		<li>To perform the tail clamp method, secure the tail of a rat from the MOD group by clamping the tail with a standard paper clip at a distance of 1-2 cm from the root of the rat&#39;s tail. Measure the clamping time for a duration of 1 min (n = 12).</li>
		<li>In the water deprivation method, withhold water from a MOD group rat by removing its water&#160;bottle and record for 24 h. 
		NOTE: The time of the start of the water deprivation was recorded so that the end time could be accurately calculated; the behavior of the rats during this period was observed, including activity, appetite and mental state.</li>
		<li>In the food deprivation method, withhold food from a MOD group rat and record for 24 h. 
		NOTE: Record the time when food deprivation begins so that the end time can be accurately calculated; ensure that the rats are adequately hydrated during this period; and observe the behavior of the rats during this period, including activity and mental status, etc.</li>
		<li>For cold stimulation at 4 &#176;C, place a rat from the MOD group in a bucket of cold water and record for 5 min. Ensure that the water temperature remains consistent throughout the test. At the conclusion of the experiment, dry the rat with a blower and return it to its original cage. 
		NOTE: Maintain the cold water temperature at 4 &#176;C using a thermometer and ice cubes to regulate the water temperature when it rises. The water should be changed regularly to ensure that the water quality is clean and the temperature is constant. When swimming, all limbs and trunks of the rats should be immersed in cold water except the head. The depth of the water must be greater than the length of the rat&#39;s body to prevent the rat from jumping out of the water due to contact with the bottom of the bucket.</li>
		<li>To administer 45 &#176;C heat stress, place a MOD group rat in an incubator and record for 5 min, ensuring that the temperature remains stable throughout the test.</li>
		<li>In the inversion of the light-dark cycle, envelop the cage in a black cloth for 1 h&#160;to simulate daytime darkness. Subsequently, illuminate the cage for 12 h during the night to mimic daylight. Record the rat&#39;s&#160;behavior, food and water intake, and sleep patterns for 24 h.</li>
		<li>For the wet padding experiment, introduce 200 mL of water into a cage containing 100 g of padding. House a MOD group rat in the wet cage and record the behavior of the rats under wet bedding stress, including activity, appetite, water intake, etc. Observe whether there are any abnormal behaviors or uncomfortable reactions, such as the skin and hair condition of the rats, which may be caused by the wet padding, and record them in time for subsequent analysis for 24 h. Following the test, dry the rat with a blower and return it to a cage with fresh wood shavings.</li>
		<li>In the cage incline method, situate a MOD group rat in a cage tilted at a 45&#176; angle against a wall and record for 24 h. Use the cage frame structure to adjust the angle and secure the cage in place. 
		NOTE: Calculate the time from start to finish and observe the behavior of the rats during the period of tilting the cage, including activity, appetite, and mental state, and ensure that the angle of the tilted cage is set correctly and remains stable to ensure the accuracy and reproducibility of the experiment.</li>
		<li>For high-speed shaking, place a MOD group rat in a mechanical shaker set to 160 rpm and record the rat for 15 min. Behavioral testing methods are subsequently utilized to evaluate the successful establishment of the model.</li>
		<li>After applying stressors, relocate the MOD group cages from the CUMS room back to the housing room. During the 4-week stress exposure period, maintain the CON group in their home cages situated in the housing room.</li>
	</ol>
	</li>
	<li>Precautions during experiments
	<ol>
		<li>Transfer cages of the MOD group back to the general housing room after applying the stressor in the CUMS room.</li>
		<li>Animal monitoring during CUMS modeling
		<ol>
			<li>During tail clamping, the animal is likely to struggle due to the induced stimulation. Throughout this period, continuously monitor the clamp. If it becomes dislodged, pause the timer, reapply the clamp, and then resume the timer for 1 min.</li>
			<li>Do not impose&#160;water deprivation and wet bedding stressors simultaneously. 
			NOTE: Avoiding the simultaneous imposition of wet padding and water deprivation helps maintain experimental integrity, reduces confounding variables, and promotes animal welfare.</li>
			<li>The animal&#39;s body temperature and ambient room temperature may raise the water temperature during cold water swimming. Therefore, adjust by adding ice water or ice cubes to maintain a constant water temperature.</li>
			<li>Observe the rats at 30 min intervals during the application of stressors, except during diurnal reversal. Pay special attention to signs of unusual distress, such as shivering, lethargy, or lack of movement. If such symptoms are observed-particularly potential hypothermia during 4 &#176;C cold water swimming and wet bedding-immediately remove the rat from the stressor. 
			NOTE: Remove the experimental animals from the study when they have&#160;health problems, such as infection, severe trauma, aggressive behaviours, abnormal mobility, etc.,The conditions for removing the animals from the study typically revolve around protecting their health and safety while ensuring the integrity of experimental outcomes.</li>
			<li>Conduct daily inspections for wounds or other physical or behavioral abnormalities in each rat. If any abnormalities are observed, consult the laboratory veterinarian to determine whether the rat should be excluded from the experiment.</li>
			<li>Weigh each rat every 3 days. If an animal loses more than 20% of its pre-feeding basal body weight, it must be excluded from the experiment.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Behavioral tests
<ol>
	<li>To begin, administer the depression stimuli in conjunction with isolation for 28 days to all rats, excluding the control group. House the rats in individual cages. Refer to Table 2 for specifics on the depression stimuli conditions.</li>
	<li>For the open field test, partition a black box into 25 square sections of equal area. Install a video tracking analysis system in the box. Place the rat in the central square and monitor its horizontal and vertical activities for a duration of 5 min. 
	NOTE: The dimensions of the box are 500 mm x 500 mm x 300 mm. Activity data will be gathered using the video tracking system to assess anxiety-related behaviors in the rodents when exposed to a novel environment.</li>
	<li>Subsequently, tally the number of squares traversed by the rat, using all paws, to quantify horizontal activity. Count instances of standing and grooming as indicators of vertical activity. After each test, sanitize the box using 75% alcohol to eliminate any residual rat odors for subsequent tests.</li>
	<li>Next, anhedonia is assessed through the sucrose preference test. Place two bottles on the cage lid: bottle A contains pure water, while bottle B contains 1% sucrose solution. Allow ad libitum access to both solutions for the rat. Weigh the bottles before and after consumption to calculate the 60 min sucrose preference rates on days 0, 7, 14, 21, and 28. The formula is as follows: 
	Sucrose consumption = <img alt="Equation 1" src="/files/ftp_upload/65889/65889eq01.jpg" style="margin: auto;" /> &#215; 100%</li>
	<li>To measure spatial memory and learning abilities, employ the Morris water maze test. Divide the pool into four quadrants, numbering them from one to four. Place a submerged resting platform 1 cm below the water surface in the third quadrant.</li>
	<li>Introduce milk to the pool to increase water opacity and maintain a water temperature of approximately 23 &#176;C throughout the experimental procedure.</li>
	<li>Place each rat in various quadrants of the maze, allowing them 120 s to locate the hidden platform. The rats must rely on spatial memory and learning skills to remember the platform&#39;s location. Once they learn the platform&#39;s location, they can swim directly to it. Record the latency time using the Morris water maze video tracking system.</li>
	<li>Position the rat at a fixed location in the pool. If the subject fails to locate the hidden platform within 120 s, record the latency as 120 s.</li>
	<li>Finally, remove the hidden platform, place the rat back into the water, and record the number of zone-crossings over a 120 s period.</li>
</ol>
3. Statistical analysis
<ol>
	<li>To assess significant differences in biochemical parameters, employ one-way analysis of variance (ANOVA) followed by Duncan&#39;s post hoc test. Present the data as mean &#177; standard error (SE) and regard a p-value of less than 0.05 as statistically significant.</li>
</ol>

Establishing and Validating a Chronic Unpredictable Mild Stress Rat Model

The authors have no conflicts of interest to disclose.

Depression is a mental disorder characterized by symptoms such as low mood, lack of pleasure, and reduced energy30. In the field of depression research, the establishment of a reliable animal model is crucial for advancing therapeutic interventions. Among various animal models, the CUMS model is particularly noteworthy for its high reliability, validity, and its congruence with the characteristics of human depression31. It is well-suited to mimic the cumulative effects of low-level stressors over an extended period across various settings. In this study, we established a CUMS-induced depression model in rats, employing a range of stressors outlined in step 1.2.3 and Table 2. Several aspects of the stress protocol are critical for the model's success. Firstly, rats in all MOD groups must be housed individually, unlike those in the CON group. Secondly, the CUMS stress regimen should be carefully planned to maintain unpredictability. Specifically, rats should be subjected to a randomized selection of seven out of the nine possible stressors on a weekly basis. The variability and unpredictability of these stressors are essential for the model's efficacy32,33. Lastly, repeated use of the same stressor should be minimized to prevent the rats from adapting, which could compromise the integrity of the experimental outcomes. Moreover, the stressor settings were designed to be adjustable throughout the course of the study.
The CUMS model offers several advantages as a depression model. It closely aligns with the pathogenesis of human depression and accurately represents most clinical symptoms and underlying mechanisms34. One of its key strengths is that it uses anhedonia, or the lack of pleasure, as a quantifiable indicator6,7. The model also accounts for the various factors that contribute to the onset of depression35,36. Importantly, the CUMS model is designed for long-term application, spanning several months. This duration is consistent with the needs of a depression model and allows for the assessment of drugs in a chronic dosing regimen, as well as the identification of compounds with rapid mechanisms of action36. Due to its comprehensive nature, the CUMS model has become widely accepted in the scientific literature.
Despite these advantages, the CUMS model is not without limitations5,37. One of the primary drawbacks is its resource-intensive nature, requiring significant investments in environment, manpower, and time, which consequently results in low reproducibility38. There are also inconsistencies in the literature regarding the selection and intensity of stressors, as well as the sequence in which they are applied38. Such inconsistencies make it challenging to replicate the model across different laboratories. Furthermore, the literature lacks a detailed time schedule for applying stressors, adding another layer of ambiguity39,40. Another issue arises from the variation in combination methods employed across different studies; these often lack explicit rationale, creating difficulties in model replication. Subject animals are highly sensitive to minor alterations in the stressor design, which also contributes to low reproducibility40. Moreover, using a limited number of stressors can lead to habituation among the subject animals, making the stressor design predictable. Finally, animals from different laboratories show varied responses to stressors, contributing to the poor reproducibility across studies. In the existing literature, Wistar Kyoto (WKY) rats have been identified as particularly susceptible to a depression-like phenotype following exposure to CUMS25,41. This suggests that inter-strain variability can significantly affect susceptibility to stress-induced behavioral changes. Similarly, the SPT, a key metric often used for assessing the efficacy of depression models, presents its own set of challenges. Specifically, individual rats show variability in their consumption of a 1% sucrose solution20,42. Under stress conditions, both stress-prone and stress-resistant animals exhibit alterations in their consumption of sweet solutions. Consequently, it becomes challenging to ascertain the proportion of animals within a given batch that are genuinely stress-resistant or belong to the opposite category. This variability may result in some animals displaying SPT outcomes that do not accurately reflect their disease state.
To enhance the reliability of the CUMS model, we propose several methodological refinements. First, it is crucial to segregate animals in the CON group from those in the MOD group to mitigate the impact of olfactory, visual, and auditory stimuli, which can induce stress in the animals43,44. Second, to minimize variability, it is advisable to designate one or two experienced laboratory personnel to conduct all experiments. This approach serves to reduce errors attributable to individual factors. In addition, prior research has identified stress-resilient test animals13; hence, it would be prudent to screen for such groups either prior to formal experimentation or post hoc to better interpret the results. Regarding the SPT test, which is a reward-based behavioral assessment that gauges intrinsic anhedonia, it is essential to determine the optimal sucrose concentration before initiating the CUMS protocol. Preliminarily establishing preference based on sucrose concentration can markedly enhance the test's discriminative sensitivity20. However, it should be noted that rats subjected to 24 h food and water deprivation may indiscriminately consume any available liquid due to extreme thirst35,45. To obtain a more accurate measure of sucrose preference, we recommend assessing consumption during the nocturnal phase of the circadian cycle, when rodents are most active. Stressors applied during the diurnal (inactive) phase could result in chronic sleep deprivation, thereby introducing an unintended stressor46,47. Finally, it is imperative to meticulously control for the influence of manageable factors described above to ensure that the data collected are as reliable as possible.
The current Western clinical treatment regimens for depression often come with pronounced toxic side effects and a high likelihood of relapse upon cessation of medication. Investigating treatment approaches rooted in traditional medicine and delving into the pathogenesis of depression through the lens of Mongolian medicine theory could offer innovative insights into the disease's etiology. In this context, the development of animal models of depression that align with traditional medicine principles stands as a pivotal step in Mongolian medicine research. The selection of the nine stressors in the solitary-culture CUMS modeling method is particularly noteworthy; it is informed by Mongolian medicine's theory of the three homors and the inherent relationship between the four auxiliary conditions and the three homors. This approach exhibits a high degree of compatibility with the unique tenets of Mongolian medicine. Phenotypic analyses of depression, in conjunction with external factors, reveal significant correlations with the Mongolian medical concepts of Wind and Phlegm (see Table 2). Specifically, wind-related dysfunctions manifest clinically as symptoms such as loss of energy, insomnia, forgetfulness, fatigue, and pain. Dysfunctions related to Phlegm, on the other hand, result in clinical manifestations like unresponsiveness, blurred consciousness, pessimism, and negativity. These symptoms align closely with the core symptoms of MDD48,49. Therefore, the aforementioned CUMS model serves as a pathological model that effectively simulates the complex interactions among external conditions and the balance of the three types of homors, as posited by Mongolian medicine theory, thereby contributing to the onset of disease. 
The strengths of this model are multifaceted. Firstly, it adheres to contemporary medical etiological theories, satisfying the criteria set for animal models within the realm of modern medicine. Secondly, it incorporates principles from traditional Mongolian medicine to replicate specific symptoms of human diseases in animals, thus embodying characteristics unique to Mongolian medical practices. Given its alignment with both modern and traditional medical theories, this model serves as an effective platform for foundational research in Mongolian medicine. Therefore, the integration of diseases and symptoms within this animal model represents a confluence of research in Mongolian and modern medicine. While the model has its limitations, its ongoing refinement will significantly contribute to the future of basic research in Mongolian medicine.
Animal models have become indispensable in the exploration of depression due to the ethical and practical challenges associated with direct human brain study. Despite their inherent limitations in fully replicating the complexities of depression, the evolution and validation of these models remain a continual process. In this study, we have combined principles from both Mongolian and modern medicine to develop an animal model of depression. This collaborative approach offers valuable insights for constructing a more comprehensive animal model that encompasses both traditional Mongolian and modern medical theories.

Major depressive disorder (MDD) is a prevalent mental illness, ranking as the third leading cause of disability globally and affecting over 300 million people1,2,3. Notably, it is estimated that at least half of the affected individuals do not receive adequate treatment4. Given this gap, animal models serve as a crucial tool for investigating the etiology of depression. To date, more than 20 different animal models for depression exist5. Among these, the chronic unpredictable mild stress (CUMS) model, refined by Paul Winer in 1987, is the most frequently utilized6. The CUMS model operates on the premise that exposing rodents to a diverse range of socio-environmental stressors leads to symptoms akin to anxiety, tension, and depression. The methodology involves exposing animals to various mild stressors over several weeks, culminating in a range of behavioral alterations, including anhedonia and depressive-like behaviors7,8. These changes are accompanied by shifts in endocrine and neurotransmitter profiles, such as a reduction in 5-HT9,10. These outcomes closely mirror those observed in humans diagnosed with MDD, thereby validating the model's utility. The CUMS model is particularly valued for its effectiveness in evaluating antidepressants, manifesting high levels of surface, structural, and predictive validity11,12. Unlike other models, CUMS is sensitive to the effects of chronic administration of monoaminergic antidepressants. For instance, selective serotonin reuptake inhibitors (SSRIs) such as citalopram, paroxetine, and fluoxetine have been shown to prevent and reverse anhedonia under conditions of chronic stress12,13. Additionally, new rapid-acting antidepressants, such as ketamine, have also demonstrated efficacy in this model14,15. In contrast, other tests like the forced swimming test (FST) and tail suspension test (TST) are less reliable for modeling long-term behavioral changes, often reflecting adaptations to acute stress rather than enduring symptoms of depression16. These characteristics underscore the robust validity of the CUMS model in depression research. One of the most salient features of the CUMS model, recognized for its high reliability in classical studies, is anhedonia-the incapacity to experience pleasure or interest in everyday activities17,18. This phenomenon is commonly evaluated using sucrose preference tests, and many antidepressants have been shown to reverse reduced sucrose consumption. Several other metrics are also commonly employed in the CUMS literature, including the open field test (OFT), which assesses voluntary motor behavior, exploratory tendencies, and tension, thereby gauging the severity of depression19. Other tests such as the elevated plus maze (EPM) evaluate anxiety-like behaviors, the Morris water maze test (MWM) examines cognitive functioning20, and the FST assesses susceptibility to negative emotions and behavioral despair20. Furthermore, the majority of stressors that impact humans are social in nature. Individuals with suboptimal social relationships, characterized by limited social activities, networks, and support, are at a heightened risk for various diseases21,22. This is also relevant in the case of rodents, who are social animals living in groups. For example, rats that are housed in isolation exhibit characteristics of what is termed isolation syndrome, which induces social stress and expedites the onset of depression23.
Mongolian medicine, a significant branch of Chinese medicine, posits that the onset of disease is a complex interplay between intrinsic and external factors. These external factors, referred to as the four auxiliary conditions, encompass climate change, diet, lifestyle, and sudden events such as infections, startling incidents, and psychological disorders. The disease process is conceptualized as an ongoing interaction between three elements-referred to as the three types of homors-and the Seven Bodily Constituents in concert with the four auxiliary conditions24. Mongolian medicine holds that the human body functions as an integrated entity, maintained by a relative balance among the three homors. A disruption in this balance is considered to be a precursor to illness24. Given the pivotal role of animal experimentation in bridging traditional and modern medicine, it is crucial to develop animal models that are relevant for research in the field of Mongolian medicine. Accordingly, we employed a 28-day isolation methodology coupled with CUMS to simulate these physiological and psychological stressors. We selected nine specific unpredictable stressors and sought to underpin this modeling method through the Three homors theory of Mongolian medicine. Establishing a robust animal model is fundamental for advancing basic research in Mongolian medicine and will contribute significantly to its foundational studies.

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chronic unpredictable mild stress in rats based on the mongolian medicine

Depression is a prevalent affective disorder and constitutes a leading cause of global disability. The limitations of current pharmacological interventions contribute to the substantial health burden attributed to this condition. There is a pressing need for a deeper understanding of the underlying mechanisms of depression, making pre-clinical models with translational potential highly valuable. Mongolian medicine, a subset of traditional medicine, posits that disease occurrence is closely tied to the equilibrium of wind, bile, and Phlegm. In this study, we introduce a protocol for the chronic unpredictable mild stress (CUMS) method in rats. Within this framework, rats are subjected to a series of fluctuating, mild stressors to induce a depression-like phenotype, mimicking the pathogenesis of human depression. Behavioral assays employed in this protocol include the sucrose preference test (SPT), indicative of anhedonia-a core symptom of depression; the open field test (OFT), which measures anxiety levels; and the Morris water maze test (MWM), which evaluates spatial memory and learning abilities. The CUMS method demonstrates the capability to induce anhedonia and to cause long-term behavioral deficits. Furthermore, this protocol is more aligned with Mongolian medical theory than other animal models designed to elicit depression-like behavior. The development of this animal model and subsequent research provide a robust foundation for future innovative studies in the realm of Mongolian medicine.

Results from the behavioral tests in the CUMS-induced rat depression model 
To corroborate the efficacy of the CUMS procedure for inducing depression-like behaviors, a manipulation check was performed. Male Sprague-Dawley (SD) rats were randomly allocated to either the MOD or CON group for a period of 4 weeks, as outlined in step 1.2.3. Subsequently, the rats were sacrificed, and their hippocampi were completely dissected for assessment of 5-HT, a neurotransmitter strongly associated with depression pathophysiology10, using enzyme-linked immunosorbent assay (ELISA)28.
Before CUMS induction, no significant differences were observed among the tested groups in the OFT score, sucrose preference, or MWM analysis.
After establishing the CUMS model, a one-way analysis of variance (ANOVA) revealed a significant impact of CUMS on rat anxiety behaviors. Specifically, in the OFT, significant differences were observed between the groups. The MOD group exhibited lower vertical and horizontal scores compared to the CON group (**P &#60; 0.01; Figure 1A,B,E), suggesting that the CUMS procedure was effective in inducing anxiety.
Subsequently, the sucrose preference test (SPT) was administered over 5 days, as described in step 2.4, to evaluate whether the rats showed signs of anhedonia post-CUMS exposure. Results indicated no significant differences in sucrose preference percentage between the groups on day 0. However, one-way ANOVA revealed a significant difference in sucrose intake between the groups on day 28. Specifically, the sucrose preference percentage in the MOD group was lower than that in the CON group (**P &#60; 0.01; Figure 1C), confirming the effectiveness of the CUMS protocol in inducing anhedonia.
A one-way ANOVA revealed that CUMS significantly impacted the spatial memory and learning abilities of rats, as assessed by the MWM test. The latency for the MOD group to locate the platform was notably longer than that for the CON group (**P &#60; 0.01). In terms of crossing times, the MOD group crossed fewer times than the CON group (**P &#60; 0.01; Figure 1D,F). These findings support the efficacy of the CUMS protocol in inducing memory impairment in rats.
Results of hippocampal 5-HT levels in the CUMS-induced rat depression model 
One-way ANOVA on hippocampal 5-HT levels indicated a significant difference among the groups (**P &#60; 0.01). The MOD group exhibited reduced hippocampal 5-HT levels when compared to the CON group (Figure 2). These results suggest that the CUMS protocol effectively decreased hippocampal 5-HT levels, a phenomenon commonly observed in human depression29.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65889/65889fig01.jpg" /> 
Figure 1: Effects of the CUMS-induced rat depression model. (A) Open field test (OFT) vertical scores. (B) Open field test horizontal scores. (C) Sucrose consumption levels (%) on days 0 and 28. CUMS-induced anhedonia and reduced sucrose preference in rats. (D) Latency time and zone crossing numbers in the MWM test. Results are presented as the mean &#177; SE (n = 12 rats per group). **P &#60; 0.01 indicates significant changes in the model (MOD) group compared to the control (CON) group calculated using one-way analysis of variance (ANOVA). (E) OFT tracks for rats in each group. (F) MWM tracks for each group of rats. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65889/65889fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65889/65889fig02.jpg" /> 
Figure 2: Effect on brain 5-HT levels in CUMS-induced depression model in rats. Rats subjected to 4 weeks of CUMS demonstrated a significant reduction in hippocampal 5-HT levels compared to the control (CON) group. Results are presented as the mean &#177; SE (n = 12 rats per group). **P &#60; 0.01 indicates significant changes in the model (MOD) group compared to the CON group calculated using one-way analysis of variance (ANOVA). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65889/65889fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Stressors</td>
			<td>Days</td>
		</tr>
		<tr>
			<td>Tail clamp 1 min</td>
			<td>1, 7, 20</td>
		</tr>
		<tr>
			<td>Water deprivation 24 h</td>
			<td>3, 18, 28</td>
		</tr>
		<tr>
			<td>Food deprivation 24 h</td>
			<td>2, 16, 25</td>
		</tr>
		<tr>
			<td>Swimming in 4 &#176;C cold water 5 min</td>
			<td>4, 11, 19, 23</td>
		</tr>
		<tr>
			<td>45 &#176;C heat stress 5 min</td>
			<td>5, 14, 17, 27</td>
		</tr>
		<tr>
			<td>Inversion of light dark cycle</td>
			<td>6, 12, 22</td>
		</tr>
		<tr>
			<td>Wet padding</td>
			<td>9, 15, 26</td>
		</tr>
		<tr>
			<td>Cage incline 24 h</td>
			<td>10, 21</td>
		</tr>
		<tr>
			<td>High-speed shaking 15 min</td>
			<td>8, 13, 24</td>
		</tr>
	</tbody>
</table>
Table 1: Chronic unpredictable mild stress (CUMS) schedule. Stressors and execution dates for the induction of CUMS in rat subjects.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Stressors</td>
			<td>Four Auxiliary Conditions</td>
			<td>Three Homors change</td>
		</tr>
		<tr>
			<td>Tail clamp 1 min</td>
			<td>Sudden factors</td>
			<td>Wind&#8593; Bile&#8593;</td>
		</tr>
		<tr>
			<td>Water deprivation 24 h</td>
			<td>Diet</td>
			<td>Wind&#8593;</td>
		</tr>
		<tr>
			<td>Food deprivation 24 h</td>
			<td>Diet</td>
			<td>Wind&#8593;</td>
		</tr>
		<tr>
			<td>Swimming in 4 &#176;C cold water 5min</td>
			<td>Living</td>
			<td>Phlegm&#8593;&#160;</td>
		</tr>
		<tr>
			<td>45 &#176;C heat stress 5min</td>
			<td>Living, Climate change</td>
			<td>Bile&#8593;</td>
		</tr>
		<tr>
			<td>Inversion of light dark cycle</td>
			<td>Living&#160;</td>
			<td>Wind&#8593;</td>
		</tr>
		<tr>
			<td>Wet padding</td>
			<td>Living</td>
			<td>Phlegm&#8593;</td>
		</tr>
		<tr>
			<td>Cage incline 24 h</td>
			<td>Living</td>
			<td>Wind&#8593;</td>
		</tr>
		<tr>
			<td>High-speed shaking 15min</td>
			<td>Sudden factors</td>
			<td>Wind&#8593;</td>
		</tr>
	</tbody>
</table>
Table 2. Nine stressors with the four auxiliary conditions and the theory of three homors in Mongolian medicine. The intrinsic relationship between the nine stressors, the theory of three homors, and four auxiliary conditions in Mongolian medicine.

Watch this Scientific Journal Video about Chronic Unpredictable Mild Stress in Rats based on the Mongolian medicine at JoVE.com

Author Spotlight: Establishing a Rodent Model for Investigating Depression Factors in Traditional Mongolian Medicine

This protocol outlines a chronic unpredictable mild stress (CUMS) model for depression based on Mongolian medical theory, along with methods for validating behavioral tests.

Chronic Unpredictable Mild Stress in Rats based on the Mongolian medicine

Depression is a prevalent affective disorder and constitutes a leading cause of global disability. The limitations of current pharmacological ...

chronic-unpredictable-mild-stress-rats-based-on-mongolian

Nanyang Technological University

Research

JoVE Journal

Medicine

802 Views.  Inner Mongolia Medical University. De reikwijdte van ons onderzoek is Mongools medisch onderzoek. In deze studie onderzoeken we de factoren die bijdragen aan depressie in de Mongoolse geneeskunde, met een bijzondere nadruk op de interactie tussen interne factoren, zoals wind, biofilm en externe factoren. Het probleem van het voldoen aan het ontbreken van traditionele Mongoolse medische modellen werd opgelost.Dit protocol richt zich op het opstellen van een diermodel op basis van de Mongoolse medische theorie en geeft ve...

Video: Auteur in de schijnwerpers: het opzetten van een knaagdiermodel voor het onderzoeken van depressiefactoren in de traditionele Mongoolse geneeskunde

Acute and Chronic Tactile Sensory Testing after Spinal Cord Injury in Rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia3. Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI4-5. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically5. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery4-5. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.

We describe two tactile sensory testing methods for acute or chronic periods of spinal cord injury in rats. These validated procedures can detect the development and maintenance of allodynia-like sensations.

Spinal cord injury (SCI) impairs sensory systems causing allodynia1-8. To identify cellular and molecular causes of allodynia, sensitive and valid sensory ...

An Unpredictable Chronic Mild Stress Protocol for Instigating Depressive Symptoms, Behavioral Changes and Negative Health Outcomes in Rodents

Chronic, unresolved stress is a major risk factor for the development of clinical depression. While many preclinical models of stress-induced depression have been reported, the unpredictable chronic mild stress (UCMS) protocol is an established translationally-relevant model for inducing behavioral symptoms commonly associated with clinical depression, such as anhedonia, altered grooming behavior, and learned helplessness in rodents. The UCMS protocol also induces physiological (e.g., hypercortisolemia, hypertension) and neurological (e.g., anhedonia, learned helplessness) changes that are clinically associated with depression. Importantly, UCMS-induced depressive symptoms can be ameliorated through chronic, but not acute, treatment with common SSRIs. As such, the UCMS protocol offers many advantages over acute stress protocols or protocols that utilize more extreme stressors. Our protocol involves randomized, daily exposures to 7 distinct stressors: damp bedding, removal of bedding, cage tilt, alteration of light/dark cycles, social stresses, shallow water bath, and predator sounds/smells. By subjecting rodents 3-4 hr daily to these mild stressors for 8 weeks, we demonstrate both significant behavioral changes and poor health outcomes to the cardiovascular system. This approach allows for in-depth interrogation of the neurological, behavioral, and physiological alterations associated with chronic stress-induced depression, as well as for testing of new potential therapeutic agents or intervention strategies.

The unpredictable chronic mild stress (UCMS) protocol is a validated method for studying behavioral and physiological changes associated with chronic stress and depressive symptoms. Eight weeks of imposition of the UCMS protocol induces behavioral changes and poor health outcomes in rodents of either gender.

Chronic, unresolved stress is a major risk factor for the development of clinical depression. While many preclinical models of stress-induced depression ...

Behavior

Chronic Constriction of the Sciatic Nerve and Pain Hypersensitivity Testing in Rats

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of the population1,2. Symptoms include spontaneous (tingling, burning, electric-shock like) pain, dysaesthesia, paraesthesia, allodynia (pain resulting from normally non-painful stimuli) and hyperalgesia (an increased response to painful stimuli). The sensory symptoms are co-morbid with behavioural disabilities, such as insomnia and depression. To study chronic neuropathic pain several animal models mimicking peripheral nerve injury have been developed, one of the most widely used is Bennett and Xie's (1988) unilateral sciatic nerve chronic constriction injury (CCI)3 (Figure 1). Here we present a method for performing CCI and testing pain hypersensitivity.
CCI is performed under anaesthesia, with the sciatic nerve on one side exposed by making a skin incision, and cutting through the connective tissue between the gluteus superficialis and biceps femoris muscles. Four chromic gut ligatures are tied loosely around the sciatic nerve at 1 mm intervals, to just occlude but not arrest epineural blood flow. The wound is closed with sutures in the muscle and staples in the skin. The animal is then allowed to recover from surgery for 24 hrs before pain hypersensitivity testing begins.
For behavioural testing, rats are placed into the testing apparatus and are allowed to habituate to the testing procedure. The area tested is the mid-plantar surface of the hindpaw (Figure 2), which falls within the sciatic nerve distribution. Mechanical withdrawal threshold is assessed by mechanically stimulating both injured and uninjured hindpaws using an electronic dynamic plantar von Frey aesthesiometer or manual von Frey hairs4. The mechanical withdrawal threshold is the maximum pressure exerted (in grams) that triggers paw withdrawal. For measurement of thermal withdrawal latency, first described by Hargreaves et al (1988), the hindpaw is exposed to a beam of radiant heat through a transparent glass surface using a plantar analgesia meter5,6. The withdrawal latency to the heat stimulus is recorded as the time for paw withdrawal in both injured and uninjured hindpaws. Following CCI, mechanical withdrawal threshold, as well as thermal withdrawal latency in the injured paw are both significantly reduced, compared to baseline measurements and the uninjured paw (Figure 3). The CCI model of peripheral nerve injury combined with pain hypersensitivity testing provides a model system to investigate the effectiveness of potential therapeutic agents to modify chronic neuropathic pain. In our laboratory, we utilise CCI alongside thermal and mechanical sensitivity of the hindpaws to investigate the role of neuro-immune interactions in the pathogenesis and treatment of neuropathic pain.

Due to the simplicity of surgery and the robust behavioural outcome, chronic constriction of the sciatic nerve is one of the pre-eminent animal models of neuropathic pain. Within 24 hrs following surgery, pain hypersensitivity is established and can be quantitatively measured using a von Frey aesthesiometer (mechanical test) and plantar analgesia meter (thermal test).

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of ...

A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these animal models. The rat is one particular model animal where an epigenetic tool can complement many pharmacological and behavioral studies to provide insightful mechanistic information. To this end, we adapted the SureSelect Target Capture System (referred to as Methyl-Seq) for the rat, which can assess DNA methylation levels across the rat genome. The rat design targeted promoters, CpG islands, island shores, and GC-rich regions from all RefSeq genes. 
To implement the platform on a rat experiment, male Sprague Dawley rats were exposed to chronic variable stress for 3 weeks, after which blood samples were collected for genomic DNA extraction. Methyl-Seq libraries were constructed from the rat DNA samples by shearing, adapter ligation, target enrichment, bisulfite conversion, and multiplexing. Libraries were sequenced on a next-generation sequencing platform and the sequenced reads were analyzed to identify DMRs between DNA of stressed and unstressed rats. Top candidate DMRs were independently validated by bisulfite pyrosequencing to confirm the robustness of the platform.
Results demonstrate that the rat Methyl-Seq platform is a useful epigenetic tool that can capture methylation changes induced by exposure to stress.

Here, we describe the protocol and implementation of Methyl-Seq, an epigenomic platform, using a rat model to identify epigenetic changes associated with chronic stress exposure. Results demonstrate that the rat Methyl-Seq platform is capable of detecting methylation differences that arise from stress exposure in rats.

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these ...

Biochemistry

The Unpredictable Chronic Mild Stress Protocol for Inducing Anhedonia in Mice

Depression is a highly prevalent and debilitating condition, only partially addressed by current pharmacotherapies. The lack of response to treatment by many patients prompts the need to develop new therapeutic alternatives and to better understand the etiology of the disorder. Pre-clinical models with translational merits are rudimentary for this task. Here we present a protocol for the unpredictable chronic mild stress (UCMS) method in mice. In this protocol, adolescent mice are chronically exposed to interchanging unpredictable mild stressors. Resembling the pathogenesis of depression in humans, stress exposure during the sensitive period of mice adolescence instigates a depressive-like phenotype evident in adulthood. UCMS can be used for screenings of antidepressants on the variety of depressive-like behaviors and neuromolecular indices. Among the more prominent tests to assess depressive-like behavior in rodents is the sucrose preference test (SPT), which reflects anhedonia (core symptom of depression). The SPT will also be presented in this protocol. The ability of UCMS to induce anhedonia, instigate long-term behavioral deficits and enable reversal of these deficits via chronic (but not acute) treatment with antidepressants strengthens the protocol&#39;s validity compared to other animal protocols for inducing depressive-like behaviors.

Here we present the unpredictable chronic mild stress protocol in mice. This protocol induces a long-term depressive-like phenotype and enables to assess the efficacy of putative antidepressants in reversing the behavioral and neuromolecular depressive-like deficits.

Depression is a highly prevalent and debilitating condition, only partially addressed by current pharmacotherapies. The lack of response to treatment by ...

Evaluating the Anti-depression Effect of Xiaoyaosan on Chronically-stressed Mice

In addition to the standardized use of antidepressant medications and psychotherapy, the usage of traditional Chinese medicine has lead to an overall improvement of patients with major depressive disorder (MDD). Therefore, the purpose of this study was to establish the mouse depressive model, observe the behavior changes associated with chronic unpredictable mild stress (CUMS), and then evaluate the anti-depression effect of Xiaoyaosan. Mice were randomly divided into four groups: a control group, a model group, a treatment group with Xiaoyaosan, and a treatment group with fluoxetine. All mice were individually kept in cages, and depression was induced in the mice by exposing them to several designed manipulations of CUMS for 21 days, as described in the protocol. Mice in the control group and model group received 0.5 mL of distilled water, while mice in the treatment groups received either Xiaoyaosan (0.25 g/kg/day) or fluoxetine (2.6 mg/kg/day). The drugs used in the study were given intragastrically daily during the entire three weeks. To estimate the depressive-like behaviors, a series of parameters including the coat state, body weight, open field test score, and sucrose preference test score were recorded. Data analysis showed that behaviors of model mice were significantly changed compared to behaviors of mice in the control group, which were improved by the treatment of Xiaoyaosan and fluoxetine. The current findings demonstrated the anti-depression effects of Xiaoyaosan on the behaviors of CUMS-induced mice and revealed that compounds from the Xiaoyaosan prescription may be worthwhile for treating depression, considering their beneficial effects on depressive-like behaviors.

Here, we present a protocol to establish the mouse depressive model, observe the behavior changes associated with chronic unpredictable mild stress (CUMS), and evaluate the anti-depression effect of Xiaoyaosan.

In addition to the standardized use of antidepressant medications and psychotherapy, the usage of traditional Chinese medicine has lead to an overall ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

Behavioral and Network Pharmacology-Based Analyses for the Traditional Mongolian Medicine Zadi-5 in a Rat Model of Depression

Zadi-5 is a traditional Mongolian medicine that is widely used for the treatment of depression and symptoms of irritation. Although the therapeutic effects of Zadi-5 against depression have been indicated in previously reported clinical studies, the identity and impact of the active pharmaceutical compounds present in the drug have not been fully elucidated.&#160;This study used network pharmacology to predict the drug composition and identify the therapeutically active compounds in Zadi-5 pills. Here, we established a rat model of chronic unpredicted mild stress (CUMS) and conducted an open field test (OFT), Morris water maze (MWM) analysis, and sucrose consumption test (SCT) to investigate the potential therapeutic efficacy of Zadi-5 in depression. This study aimed to demonstrate Zadi-5&#39;s therapeutic effects for depression and predict the critical pathway of the action of Zadi-5 against the disorder. The vertical and horizontal scores (OFT), SCT, and zone crossing numbers of the fluoxetine (positive control) and Zadi-5 groups were significantly higher (P &#60; 0.05) than those of the CUMS group rats without treatment. According to the results of network pharmacology analysis, the PI3K-AKT pathway was found to be essential for the antidepressant effect of Zadi-5.

The present protocol describes a method for the behavioral test validation and bioinformatical prediction of the therapeutic efficacy of Zadi-5, a traditional Mongolian medicine, in depression.

Zadi-5 is a traditional Mongolian medicine that is widely used for the treatment of depression and symptoms of irritation. Although the therapeutic ...

Impact of Restraint Stress on Physiology and Neurobiology in Rodent Models

Restraint to Induce Stress in Mice and Rats

Across all animal species, exposure to stressful conditions induces stress responses. One method to study the effects of stress using rodent models is the restraint stress procedure. Restraint stress has been used for decades to investigate changes in physiology, genetics, neurobiology, immunology, and other systems impacted by stress. Due to the ease of performing the procedure, low cost, and numerous modifications to scale for the intensity and duration of stress exposure, a vast literature of studies has used restraint stress in mice and rats. As one example, this study presents previously published data showing the impact of restraint stress in transgenic mice on plasma corticosterone levels and optogenetically-induced norepinephrine release. Acute restraint stress increased plasma corticosterone levels, yet this effect was blunted in mice following repeat restraint stress. However, stimulated norepinephrine release in the bed nucleus of the stria terminalis was increased only in the repeat restraint stress group. These data highlight important considerations of restraint parameters on dependent measures. Additional descriptions of restraint stress in rats are also included for comparison. Finally, the influence of the parameters of the restraint (e.g., acute vs. chronic) and characteristics of the animal subjects (strain, sex, age) are discussed.

This article covers the procedures to induce stress responses using physical restraint stress in mice and rats. Additional considerations that should be observed when selecting and using restraint stress in rodent models are discussed.

Across all animal species, exposure to stressful conditions induces stress responses. One method to study the effects of stress using rodent models is the ...

Chronic Unpredictable Mild Stress in Rats based on the Mongolian medicine

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